The effector-triggered immunity landscape of tomato against Pseudomonas syringae

Tomato (Solanum lycopersicum) is one of the world’s most important food crops, and as such, its production needs to be protected from infectious diseases that can significantly reduce yield and quality. Here, we survey the effector-triggered immunity (ETI) landscape of tomato against the bacterial pathogen Pseudomonas syringae. We perform comprehensive ETI screens in five cultivated tomato varieties and two wild relatives, as well as an immunodiversity screen on a collection of 149 tomato varieties that includes both wild and cultivated varieties. The screens reveal a tomato ETI landscape that is more limited than what was previously found in the model plant Arabidopsis thaliana. We also demonstrate that ETI eliciting effectors can protect tomato against P. syringae infection when the effector is delivered by a non-virulent strain either prior to or simultaneously with a virulent strain. Overall, our findings provide a snapshot of the ETI landscape of tomatoes and demonstrate that ETI can be used as a biocontrol treatment to protect crop plants.

Reviewer #1 (Remarks to the Author): In the paper entitled"The Effector-Triggered Immunity Landscape of Tomato against Pseudomonas syringae", the authors focused on tomato-Pseudomonas syringae interaction, aiming to unravel the ETI landscape in tomatoes.They used a comprehensive collection of P. syringae type III effectors called PsyTEC to investigate effector-triggered immunity (ETI) in various tomato varieties and cherry tomatoes.They found that tomatoes have a more limited ETI landscape compared to the model plant Arabidopsis thaliana.Only six effector families in P. syringae were capable of eliciting strong ETI responses in tomatoes.They also investigated whether ETI could be used to prime tomato plants' immunity against future infections.They discovered that ETI elicitors effectively protected tomato plants against P. syringae infection when delivered by a non-virulent strain either before or simultaneously with a virulent strain.In summary, this research provides valuable insights into the ETI landscape of tomato, revealing its distinct nature compared to Arabidopsis thaliana.Additionally, it demonstrates the potential of using ETI as a biocontrol strategy to enhance the protection of tomato crops against P. syringae infections.I think this research provides important and novel insights for the research field of plant immunity.However, the manuscript can be improved by the revision based on the comments below.

Major points
Line 421-425: In considering the potential utilization of ETI as a biocontrol strategy, it is essential to take into account the associated costs.ETI activation often leads to strong immune responses, culminating in a hypersensitive response (HR) and severe growth stunting in plants.Consequently, the concentration of the ETI-eliciting strain becomes a critical factor in achieving efficient pathogen protection while minimizing adverse effects on crop yield.It would be informative to investigate the concentration dependency of the ETI-eliciting strain on both pathogen protection and host plant growth.
Line 421-425: It is worth noting that tomatoes, which originated in the Andes Mountains and have spread worldwide relatively recently through human cultivation, may face unique challenges in dealing with Pseudomonas species as potential pathogens.It is conceivable that Pseudomonas species could be relatively new threats to tomatoes, and tomatoes might not have had sufficient time to evolve a comprehensive set of NLRs for recognizing them.In contrast, the interaction between Arabidopsis and Pseudomonas might have deeper historical roots, possibly extending further back in time.Consequently, Arabidopsis could have a more extensive repertoire of NLRs as a result of a longer co-evolutionary history with Pseudomonas.To better understand this dynamic, it would be intriguing to delve into the history of tomatoes, including their domestication timeline and their interactions with Pseudomonas species.Additionally, comparing the abundance and diversity of Pseudomonas species in the Andes Mountains, the tomato's place of origin, with other regions could provide valuable insights.Such a comparative analysis might shed light on whether the unique evolutionary history of tomatoes in the Andes has influenced their immune responses and NLR repertoire in response to Pseudomonas infections.Importantly, this perspective could help explain why wild tomatoes may not exhibit a wider recognition range of Pseudomonas effectors compared to cultivated tomatoes, highlighting the complex interplay between evolution, pathogens, and plant immune responses.
Line 418-420: There has been work on characterizing the effects of ETI-preactivation (or priming) leading to enhanced activation of PTI, and that PTI is required for ETI-induced resistance against Pst DC3000.Simultaneous activation of ETI and DC3000 infection leads to more robust immune responses (or less immunosuppression of immune responses by DC3000) in Arabidopsis thaliana.I believe the authors should mention these works in their discussion (https://doi.org/10.1038/s41586-021-03315-7and https://doi.org/10.1038/s41586-021-03316-6).
Line 95-96: 'the use of ETI as a targeted immunostimulant for priming has yet to be established'.This is not true.It has been shown that the pre-activation of ETI (using an inducible effector expression system) can prime Arabidopsis thaliana against DC3000 infection.Please cite the following work: https://doi.org/10.1093/jxb/erz571Reviewer #2 (Remarks to the Author): Lonjon et al report on a large-scale analysis of possible effector recognition events leading to immunity in tomato (cultivated S. lycopersicum).Here, the authors leverage the previously wellbuilt collection/library of Pseudomonas syringae effectors to investigate the potential of the tomato immune system to detect effector from this important bacterial pathogen.The P. syringae effector compendium (PsyTEC) was tested in Arabidopsis revealing the wide potential for effector-triggered immunity (ETI) in this model host.In the present study, using a similar high-throughput method to investigate ETI (evaluation of disease symptoms via automatized measurement of decline in greenness), the authors describe a narrower "ETI landscape" where only 6 (including 5 yet unknown) families of P. syringae effectors elicit ETI in tomato.Based on their assessment of the conservation of this ETI profile across tens of cultivars and two closely related species (S. arcanum and S. pimpinellifolium), the authors thoroughly discuss the difference observed between tomato and Arabidopsis, including a possible loss during breeding, the presence of ETI-suppressing effectors in the strains used for the screen and the limitations due to the screening method.In combination with their evaluation of effector family conservation across P. syringae species complex, the authors provide support and rationale for developing a new ETI-based protection methodology.Most of these currently rely on the identification/cloning/transfer of resistance loci mediating the effector recognition.New sources of resistance to bacterial speck disease are indeed identified in this work, although they remain to be characterized.More interestingly, the authors suggest and bring evidence that treatment (through spray-inoculation) with a strain carrying an ETI-eliciting effector prior to or concomitant with pathogenic strain infection protects tomato from bacterial speck disease.
The authors use a powerful approach to interrogate the immune potential of an important crop.Appropriate methods are used, and their limitations clearly stated.The findings from the screen are validated through alternative approaches (i.e. the decline in greenness as a proxy for disease symptoms evaluation is confirmed by bacterial enumeration in planta and ability of the identified effectors to trigger the hypersensitive response when delivered by the effectorless strain D36E).Some effector features (i.e.catalytic activity) required for recognition are further investigated and the possible nature of the resistance loci discussed.I commend the authors' efforts to provide representative raw data for the screen in different accessions.The manuscript is dense as it covers several levels of investigation (from an evolutionary perspective on both plant and pathogen to the molecular features of the recognition events).Nonetheless, this report is timely and of importance for a broad readership.
A few suggestions for improvement: Table 1: add footnote for nRecPD (indication of HGT) Table 2: add footnote for frequency parameters (e.g.number of strains examined) Table 3: change "stains" to strains (in the second column) Fig. 1 title: tomato ETI response to PsyTEC alleles (to replace "tomato response of PsyTEC") Fig. 1: axes title?What is the variability of PtoDC3000(EV) growth?Formula for data normalization?Fig. 7a: change "not expresses" to not expressed L26: eliciting strong what?L113: 21 families for ETI in Arabido, 19 families mentioned on L79 L137: change PtoDC000 to PtoDC3000 L138: add "Glamour" or "tomato" before plants L143: how is the disease score distribution analyzed?L154/252: add reference to Laflamme's study L156: remove "." after "variation in.ETI" L178: "high throughput screen", is it carried on 10-day-old tomato seedlings?L218: revise the vague subheading -effector features required for ETI elicitation?L288: quantification of "high degree" for horizontal gene transfer for HopAR1?L342: reference for lack of RPS5 homolog in tomato?L354: citing Fig. S12 here rather than Fig. 5 L414: not really/only "priming", change subheading in result section?L420: "spatial structure in local acquired resistance".It would be worth to briefly develop this to clarify this point of discussion.L433: 1M MgSO4 is the final concentration in the medium?Effector expression L434-5, L463, L467: add space between number and unit.L439: references for PtoD36E and Pto delta avrPto delta avrPtoB strains?L458: correct "umol/m2s".L459: spray inoculated or sprayed with bacterial suspensions.L467: bacterial suspension not solution L471: change to bacteria counts?(instead of "concentrations") L474: infiltrated into what?L480: change incubated to incubating.L487: add detection method/reagents.Fig. S13 legend: lines 958-961 belong to the result section.
Please find below our responses to all comments from the two reviewers of our manuscript entitled "The Effector-Triggered Immunity Landscape of Tomato against Pseudomonas syringae".We thank the reviewers for their positive feedback and constructive recommendations.In response, we have addressed all concerns below and edited the manuscript accordingly.We have also added an author Yan Lai, who performed the requested experiments and finalized the manuscript revisions.We appreciate the reviewer's suggestion to investigate the concentration dependency of the ETI-eliciting strain, and we have conducted experiments to address this aspect.We added in the result section and created figure S14 and have added the following statements to the text: "In order to investigate the dose dependency of the ETI-induced protection against PtoDC3000, we conducted co-inoculation experiments with varying concentrations of D36E::HopAB1n (OD600 1.00, 0.33, 0.10, 0.033, and 0.01).Our results revealed that ETIinduced protection was dose-dependent.A robust protective effect was consistently observed against PtoDC3000 when D36E::HopAB1n was inoculated at the higher concentrations of OD600 1.0 and 0.33 (Fig. S14a), whereas no significant protection was observed at the lower concentrations of 0.1, 0.01 and 0.033 (Fig. S14a)." As requested, we have also assessed the fitness cost of ETI-induction using plant fresh and dry-weight as a fitness proxy.These results are presented in Fig S14b,c and the following was added to the text: "To assess any potential fitness costs associated with D36E-induced ETI, we sprayed 4week old tomato plants with D36E::HopAB1n at OD600 1.00, 0.33, 0.10, 0.033, and 0.01 and measured the fresh and dry weight of the tomato plants 3 weeks post-inoculation.Our results indicate that there is no growth deficit associated with D36E::HopAB1n infection at any concentration tested as the fresh and dry weights of inoculated plants were comparable to control plants sprayed with MgSO4 (Fig. S14b,c)."We thank the reviewer for these thoughtful insights that will make for very interesting follow-up studies.As a teaser we have added the following to the Discussion: "It is possible that tomatoes have a shorter co-evolutionary history with P. syringae than Arabidopsis resulting in a more limited resistance profile against this pathogen.Probing the diversity of P. syringae species in the Andes Mountains, where tomato originated, relative to areas of domestication may shed light on the evolutionary pressures that have shaped the current ETI landscape of tomato (Peralta, .E. & D.M. Spooner.2000 ; PMID:31912142)." Line 418-420: There has been work on characterizing the effects of ETIpreactivation (or priming) leading to enhanced activation of PTI, and that PTI is required for ETI-induced resistance against Pst DC3000.Simultaneous activation of ETI and DC3000 infection leads to more robust immune responses (or less immunosuppression of immune responses by DC3000) in Arabidopsis thaliana.I believe the authors should mention these works in their discussion (https://doi.org/10.1038/s41586-021-03315-7and https://doi.org/10.1038/s41586-021-03316-6).
We again thank the Reviewer for the thoughtful comment and have added the following to the Discussion: "Furthermore, it is possible that ETI-mediated protection involves the Pattern-Triggered Immune (PTI) response, since these two branches of the plant immune system have been demonstrated to mutually potentiate one another (Ngou et al. 2021, Yuan et al., 2021;PMID: 33692545;PMID: 33692546)."Line 95-96: 'the use of ETI as a targeted immunostimulant for priming has yet to be established'.This is not true.It has been shown that the pre-activation of ETI (using an inducible effector expression system) can prime Arabidopsis thaliana against DC3000 infection.Please cite the following work: https://doi.org/10.1093/jxb/erz571 Thank you, we have included this reference to the Introduction: "It has been demonstrated that pre-activation of ETI, utilizing an inducible effector expression system, can enhance priming against PtoDC3000 infection in A. thaliana (Ngou et al;, 2020).However the effectiveness of using microbially-induced ETI as a targeted immunostimulant for priming in tomato has not yet been established."

Corrected
Reviewer #2 (Remarks to the Author): A few suggestions for improvement:  We have clarified this in the legend of Figure 1 : "Data represent the normalized results of twelve experiments.The minimum growth observed for the EV strain was 6.72 log CFU/cm 2 , the maximum was 7.32 log CFU/cm 2 , with an average of 7.12 log CFU/cm 2 .For normalization within a single experiment, the growth of a particular replicate was divided by the average growth of the empty vector strain within that specific experiment."We changed to "electing a strong immunity" L113: 21 families for ETI in Arabido, 19 families mentioned on L79 19 families were identified when the PtoDC3000 strains was used for screening, 2 more families were identified in the strain PmaES4326 (see references Laflamme et al., 2020 andMartel et al., 2022).This has been clarified in the Introduction: "Systematic analyses of the ETI landscape of the model plant A. thaliana ecotype Col-0 against P. syringae identified 69 alleles from 21 families that can trigger ETI 23, 24 ."L156: remove "." after "variation in.ETI" Removed L178: "high throughput screen", is it carried on 10-day-old tomato seedlings?We modified: "We next comprehensively screened PsyTEC for ETI in the two wild tomato species using four week-old seedlings (Fig. 3b,c S5, S6, Table 1)."

L218: revise the vague subheading -effector features required for ETI elicitation?
We modified the subheading to read: "Effector residues required for ETI and Hypersensitive Response elicitation" L288: quantification of "high degree" for horizontal gene transfer for HopAR1?This is quantified using a RecPD value as published in Bundalovic-Torma C, Desveaux D, Guttman DS.RecPD: A Recombination-aware measure of phylogenetic diversity.PLoS Comput Biol 18, e1009899 (2022)."nRecPD values range from 0.0 to 1.0, with the former reflecting genes evolving primarily through horizontal evolutionary process, and the latter reflecting genes evolving primarily through vertical descent 41 .…..This is clarified in the text : "HopAR1g, HopAR1b, HopAR1a, HopAA1i, and HopBC1b, exhibit low nRecPD values (<0.3), indicating a high degree of horizontal transfer among P. syringae isolates."

L342: reference for lack of RPS5 homolog in tomato?
We added the reference (PMID: 24885638) L414: not really/only "priming", change subheading in result section?
We changed to "ETI protects tomatoes against concomitant or subsequent P. syringae infection" L420: "spatial structure in local acquired resistance".It would be worth to briefly develop this to clarify this point of discussion.We added "This finding may be due to spatial ETI structure described as localized acquired resistance 58 , where the ETI response occurs in a 2 mm area surrounding cells in contact with the elicitor 58 .This spatial structure implies that the primary protective impact of ETI may be localized to the initial infection site." "eliciting strong", do you mean strong immunity?It would help the readers to navigate the article by stating exactly which figure they should be looking at (for example, Fig 4b-c instead of just Fig 4).

Reviewer
considering the potential utilization of ETI as a biocontrol strategy, it is essential to take into account the associated costs.ETI activation often leads to strong immune responses, culminating in a hypersensitive response (HR) and severe growth stunting in plants.Consequently, the concentration of the ETIeliciting strain becomes a critical factor in achieving efficient pathogen protection while minimizing adverse effects on crop yield.It would be informative to investigate the concentration dependency of the ETI-eliciting strain on both pathogen protection and host plant growth.

Line 421- 425 :
It is worth noting that tomatoes, which originated in the Andes Mountains and have spread worldwide relatively recently through human cultivation, may face unique challenges in dealing with Pseudomonas species as potential pathogens.It is conceivable that Pseudomonas species could be relatively new threats to tomatoes, and tomatoes might not have had sufficient time to evolve a comprehensive set of NLRs for recognizing them.In contrast, the interaction between Arabidopsis and Pseudomonas might have deeper historical roots, possibly extending further back in time.Consequently, Arabidopsis could have a more extensive repertoire of NLRs as a result of a longer co-evolutionary history with Pseudomonas.To better understand this dynamic, it would be intriguing to delve into the history of tomatoes, including their domestication timeline and their interactions with Pseudomonas species.Additionally, comparing the abundance and diversity of Pseudomonas species in the Andes Mountains, the tomato's place of origin, with other regions could provide valuable insights.Such a comparative analysis might shed light on whether the unique evolutionary history of tomatoes in the Andes has influenced their immune responses and NLR repertoire in response to Pseudomonas infections.Importantly, this perspective could help explain why wild tomatoes may not exhibit a wider recognition range of Pseudomonas effectors compared to cultivated tomatoes, highlighting the complex interplay between evolution, pathogens, and plant immune responses.
Minor pointsLine 26: "eliciting strong", do you mean strong immunity?Corrected It would help the readers to navigate the article by stating exactly which figure they should be looking at (for example, Fig 4b-c instead of just Fig 4).

Fig
Fig. 7a: change "not expresses" to not expressed Corrected is the disease score distribution analyzed?This has been clarified in the legend of Figure 2 "Distribution of disease scores (% green decline values) from the tomato var.Glamour PsyTEC screen (this study) (orange bars) and of the disease score values from the A. thaliana Col-0 PsyTEC screen 23 (green bars).The y-axis represents the number of effectors in each category when screened for ETI on the corresponding plant species and is presented as a log-scale."L154/252: add reference to Laflamme's study We added the reference.
Fig. S13 legend: lines 958-961 belong to the result section.This was removed from the Figure Legend since it was redundant with the Results section.

Table 1 : add footnote for nRecPD (indication of HGT)
We have added the footnote

Table 2 : add footnote for frequency parameters (e.g. number of strains examined)
We have added a footnote